Key words Burn injury, Me Re Sa selective medium, S.aureus, Polymerase Chain Reaction, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry, Methicillin Resistance Staphylococcus aureus.
Introduction
Staphylococcus aureus is a major pathogen associated with hospital and community acquired infections around the world (35). Before availability of antibiotics, invasive infections caused by S. aureus were often fatal (35). However, introduction of penicillin greatly improved the prognosis for patients with severe staphylococcal infections, but after a few years of clinical use, resistance appeared owing to the production of β-lactamases (25). Methicillin was designed to have resistance towards β-lactamase , but Methicillin resistant S.aureus (MRSA) strains that were resistant to β-lactam antibiotics was identified soon after methicillin introduced into clinical practices (8) and which was predominant in hospital acquired infections and community-acquired infections as well (1). In addition, according to Robinson and Enright (37) increase in number of bacterial strains showing resistance to methicillin or oxacillin has become a serious clinical and epidemiological problem for several reasons.
In clinical practices infection was a major drawback and infection related mortality was encountered in burn patients (22). Thus, measures to prevent and treat infections were essential for the survival of patients with extensive burns (20, 40, 50). Colonization of MRSA in burn tissues was associated with increased transmission of MRSA to non-burn patients and also with loss of skin grafts and delayed wound healing (15, 31) which is due to higher percentage of infection (7) .
In order to avoid mortality in burn patients, accuracy and promptness in detection of methicillin resistance of S. aureus for suitable antibiotic therapy for patients infected with these strains was of high demand and which prevent the spread of MRSA isolates in hospital environments. According to Chambers (5), strains that were oxacillin and methicillin-resistant, historically termed as MRSA, although methicillin was no longer the agent of choice for testing or treatment and was resistant to all β-lactam agents, including penicillins, cephalosporins, carbapenems, and beta lactamase inhibitor combinations. MRSA strains harbor the mec A gene, which encodes a modified PBP2 protein (PBP2N or PBP2a) with low affinity for methicillin and all β-lactam antibiotics. Further, rapid and accurate microbial identification was critical in regulating bioprocessing operations and diagnosing diseases. Efforts to develop procedures for speedy detection and specific identification of pathogenic microorganisms in samples have been actively pursued for several years (24).
The major problem associated with MRSA infection lies in identification of strains. The delay in process of identification worsens the situation (13). With reference to the detection of methicillin resistance in S.aureus, several methods (5, 45) (28, 42) including classic methods of determining minimum inhibitory concentrations (MIC), screening techniques with solid culture medium containing oxacillin or cefoxitin (47) and methods that detect mec A gene or its protein product (PBP 2 protein) (23) (4) (47) were in reports. Furthermore, identification using whole cell protein profiles using Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI TOF-MS) was also found advisable (2, 14). Since, a few reports were available on the detection of MRSA from clinical samples of burn patients and hence, it was necessary to examine and authenticate the feasibility and reliability of methods of identification of MRSA strains from burn patients.
Thus, to facilitate, ease and to authenticate MRSA strains form samples obtained from burn patients, two different test methods were attempted, viz., PCR based nuc and mec A gene identification and MALDI-TOF-MS. In brief, present approach involves isolation of microbes from external wound sites of burn patients from various hospitals in Chennai, Tamil Nadu. Identification/differentiation and characterization of Methicillin Resistant S.aureus was made using regular microbial methods (phenotypic and genotypic) and also using Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results were compared and authenticated.
Materials and methods
Collection of specimens
Burn wound infected samples were collected using sterile swabs from external wound sites of burn patients from hospital wards. A total of 106 samples were collected, labeled properly and transported to the Microbiology laboratory of Central Leather Research Institute, Chennai within 2-3 h of collection using a cooler packed with ice packs. Swabs were cultured in Nutrient broth medium for 24 h at 37°C. The cultures were then serially diluted and spread on to Nutrient agar medium (Hi Media, India).The plates were then incubated at 37°C for 24 h.
Identification and phenotypic study of bacterial strains
About thirty six bacterial strains of hospital origin obtained from Nutrient agar plates from the above experiment and four S. aureus type strains obtained from MTCC (Microbial Type Culture Collection, Chandigarh, India) were used for identification. S. epidermidis (MTCC 3382, MTCC 6810) was used as negative control in PCR amplification. E.coli (MTCC 521) was used as negative control for biochemical tests. The strains were identified based on growth characteristics on Barid Parker Agar Base with Egg Yolk Tellurite Emulsion (Hi Media, India), Gram staining, 3% potassium hydroxide (KOH) test and catalase test (18). In addition, all isolates were spot inoculated on Mueller-Hinton agar (Hi Media, India) containing 1 % of Methicillin (Sigma Chemical Co., St. Louis, Mo.) according to CLSI (formerly NCCLS) to assess the growth of the isolate (21). Further in order to identify the strains with methicillin resistance, cultures obtained were grown on Me Re Sa selective agar medium (HiMedia, India) with 1% methicillin (HiMedia, India) and incubated at 37°C for 24 h. To observe the morphological features of microbes isolated, Scanning Electron Microscopy was attempted as per procedure (6) using Jeol 840 Scanning Electron Microscope. The isolated cultures were further grown in nutrient broth medium and maintained in nutrient agar slants as well as in 10% glycerol at -20°C until further use.
Preparation of chromosomal DNA for PCR (genotypic identification)
Genomic DNA from the microbial culture was prepared as reported (36) with a few modifications. Cells from overnight culture of Mueller-Hinton broth was collected by centrifugation and suspended in 1ml of phosphate buffer (pH 7.2 ± 0.2) containing 1 mg of lysozyme and 10 µg of RNAse and incubated at 37°C for 30 min. After incubation equal volumes of Tris saturated phenol was added and DNA was extracted with equal volumes of chloroform: iso amyl alcohol and precipitated with equal volumes of ethanol and air dried for 20-30 min and finally redissolved in Tirs-EDTA buffer. Quantification of DNA was made using UV-Visible spectrophotometer (UV 2450, UV -Vis spectrophotometer, Shimadzu) at 260 nm. DNA samples were stored at 4°C until further use.
PCR for mec A and nuc gene detection
Polymerase Chain Reaction (PCR) was performed using extracted genomic DNA for simultaneous detection of nuc (3) and mec A (34). The nuc gene was responsible for production of thermostable nuclease and was included in PCR assay in order to confirm that the isolates were indeed S. aureus. PCR was performed using 12.5 µl of 1X Go Taq Green Master Mix (Promega, Madison, WI), 2.5 µl of each primer and 2 µl of DNA template to a final volume of 25 µl. Thermo cycling conditions in Eppendorf Master Cycle Gradient (Hamburg, Germany) thermocycler were as follows: initial denaturation at 94°C for 3 min followed by 30 cycles of 94°C for 1.5 min, 55°C for 2 min and 72°C for 3 min and a final extension of 10 min at 72°C. The primer sequences for nuc and mecA were shown in Table 1. The control organisms include Staphylococcus aureus MTCC 3610, Staphylococcus aureus MTCC 7443, Staphylococcus epidermidis MTCC 6810 and Staphylococcus epidermidis MTCC 3382. The primers will produce a 533 bp sequence for the mec A gene and 278 bp for the nuc gene as described (32). Ten µl of PCR product was then analyzed by agarose gel electrophoresis [2% agarose prepared in TAE (1mM EDTA/40 mM Tris acetate, pH 8.0)] at 50 V for 60 min. Gels were stained with ethidium bromide and photographed under ultraviolet light.
Sample preparation and analysis for MALDI-TOD MS of intact bacterial cells
α-cyano-4-hydroxycinnamic acid was dissolved in a 2:1 (H2O: ACN) solution containing 0.1% TFA for the preparation of saturated matrix (11). The matrix solution was filtered using a 0.5 µm filter (Millipore, India). To minimize the cross contamination in subsequent steps, a droplet of this solution was placed on parafilm with a disposable tip. Bacterial samples were then removed as colonies from agar surface with a disposable glass pipette. The tip of glass pipette containing bacteria was placed in the center of the matrix droplet allowing capillary action to draw matrix into pipette. One µl of bacterial suspension was placed on a sample pin. Duplicate samples were allowed to air dry and pins were inserted into a sample carousel, and carousel was introduced into mass spectrometer ion source.
Spectra were obtained using a Voyager-DE PRO Biospectrometry Workstation MALDI-TOF MS in positive-ion mode with an acceleration voltage of 26 kV. A pulsed nitrogen laser of 337nm was used (maximum firing rate, 20 Hz; maximum pulse energy, 300 íJ) for MALDI TOF- MS studies. Mass spectrums was collected in positive and negative modes and each spectrum was the sum of ions obtained from 100 laser shots performed in five different regions of same well. The spectra were analyzed in m/z range of 100-7000 KDa. The analysis was performed with Powerful Voyager Version 5 Software with Data Explorer software and calibrated with Bovine serum albumin as an external standard. Only peaks with intensities 0.4 mV after baseline subtraction was considered in analysis. The presence and absence of peaks were considered as fingerprints for particular isolate. Results were transferred to excel spreadsheets and analyzed using Origin Pro statistical software version 8.0.
Antibiotic susceptibility tests
The disk diffusion assay was performed according to Clinical and Laboratory Standards Institute (CLSI) (formerly known as NCCLS) guidelines. Antibiotic disks were placed on Muller Hinton agar plates pre seeded with 0.5 Mc Farland Standardized overnight cultures. Plates were then incubated at 37°C for 18 to 24 h. Experiments were done in triplicates and the zone of inhibition was measured using antibiotic zone scale (Hi-Media, India) and antibacterial activity expressed in terms of inhibition zone (mm).
Results
The isolation and intensive identification procedures, viz., gram -positive cocci; catalase positive; positive KOH test and growth on Barid Parker Agar Base with Egg Yolk Tellurite Emulsion (HiMedia, India) without supplementation of methicillin (9) provided 10 microbial isolates as S.aureus (S1-S10) from 106 swab samples collected from burn patients of >40% burn injury. Further, cultures showing denim blue appearance in Me Re Sa selective agar medium (Fig.1a) indicates MRSA as only colonies resistant to methicillin can survive in the medium. In addition, all the screened MRSA isolates grew in methicillin spot inoculation test. Fig. 1b illustrates the morphological features observed on scanning electron microscopy and found spherical shaped colonies in clusters. E.coli (MTCC 521) used in the present study as negative control exhibited negative results in biochemical tests.
Genotypic and phenotypic expression for methicillin resistance in the isolated clinical strains of S.aureus was examined using PCR. All isolates tested display positive for nuc gene confirmed S.aureus and further the presence of mecA gene in seven isolates (S1-S7) (Fig. 2) confirmed as MRSA. Negative control strains does not contain mecA gene.
Followed by PCR analysis, strains confirmed as MRSA were further subjected to MALDI-TOF-MS analysis for additional confirmation and determined for representative mass between 700-1000 KDa. Fig.3a-3d illustrates MALDI-TOF-MS spectrum of bacterial species obtained from burn wound site. With the current experimental conditions most spectral peaks were observed in the representative mass range and can be compared with earlier reports (14, 48). Fingerprint observations of S.aureus strains studied in the present study showed three common peaks with m/z of 5514, 6013 and 6587 respectively, which were observed in all S.aureus isolates and peaks of m/z 1729, 2288, 5514, 6004, 6576 and 6885 found only in MRSA strains isolated from burn wound site. Peak profile for all the isolates were summarized in Table 2. Over 90% of the peaks were reproduced in all replicates and relative intensities (but not absolute intensities) were closely comparable.
The results of the susceptibility tests for cultures obtained from clinical samples and type strains of S.aureus against 10 antibiotics were summarized in Table 3. Antibiotic inhibition zone diameters against the strains were in compliance with CLSI (formerly NCCLS) guidelines. The type strains were susceptible towards β-lactum antibiotics (oxacillin, amoxicillin and ampicillin) whereas, MRSA strains obtained from clinical samples showed resistance towards β-lactum antibiotics, gentamicin, oflaxin, streptomycin and tetracycline. However, S1 and S5 were exceptional by exhibiting resistance to ampicillin but not to oxacillin and amoxicillin. All the other isolates display resistance towards gentamicin, oflaxin, streptomycin and tetracycline except S2 and S3 which were sensitive to chloramphinicol and erythromycin.
Discussion
Staphylococcus aureus was a major cause of both healthcare associated and community acquired infections throughout the world (35). Recently, emergence of methicillin-resistant Staphylococcus aureus (MRSA) dominates and needs special attention (46). MRSA was first reported in 1961, two years after the introduction of methicillin for treatment of penicillin-resistant Staphylococcus aureus infections (16). Despite extensive infection control efforts, methicillin resistance among isolates of S.aureus has steadily increased (35).
Susceptibility testing of methicillin resistance in Staphylococcus aureus may be problematic because of the heterogeneous resistance displayed by clinical isolates (39). While methods of susceptibility testing were standardized (NCCLS), a few isolates that have been found to contain mecA yet appear to be phenotypically susceptible and have the potential to become highly resistant if exposed to anti staphylococcal penicillin's.
Oxacillin has been used to replace methicillin since it was no longer the agent of choice for treatment of S. aureus infections. Detection of mecA gene by PCR was considered to be the "gold standard method" because of its simplicity and reproducibility (38, 41). In the present study, out of 106 samples isolates tested, 10 strains were found to be S.aureus based on gram staining, morphological features and 3% KOH test and growth in Me Re Sa agar supplemented with 1% methicillin. And methicillin spot agar test further confirmed 7 of the 10 isolates as MRSA.
PCR results demonstrate 10 out of 106 isolates contain nuc gene and the presence of nuc gene was in complete agreement with the species determinations of S.aureus (17). These strains were further tested for their methicillin resistance using PCR and seven strains had mecA gene which confirms their methicillin resistance. The result from PCR amplification was in agreement with growth in Me Re Sa agar supplemented with 1% methicillin and methicillin spot inoculation tests used in this study. The detection of both genes in an isolate would confirm the identity of an isolate as MRSA(33). Tan (44) claims that conventional phenotypic methods of detecting methicillin resistance in S.aureus was difficult, but we found no dissimilarities in the present study.
According to Liang et al. (27) direct analysis of proteins from bacterial cell extracts by MALDI -TOF MS has been a potential means for the identification of bacteria. Several researchers have obtained the protein profiles of several bacterial species using MALDI (2, 14, 29). These protein profiles were distinctly different and allow differentiation of bacteria to the species level. Thus, in the present study the bacterial samples obtained from PCR study were further reconfirmed using MALDI -TOF MS and the mass spectral data was compared with reported literature (2). The spectral profiles of MRSA and S.aureus exhibited the differences in their mass range of 100 -7000 KDa under the experimental conditions as illustrated in the fig.3a-3d. At a finer level of discrimination, particularly in the peak pattern differences between MRSA and S.aureus isolates can be observed within m/z ranges of 1000-2500, 5000-5500, 6000-6500 KDa.
In the present study, resistance observed for MRSA strains isolated from burn wound site was on par with previous reports (10, 12, 30, 43). Few researchers also observed the presence of non resistant MRSA strains associated with CAMRSA (49). However, isolates S8 and S10, showed resistance against ampicillin, amoxicillin and tetracycline and this observation was supported by the previous observations of Grub et al. (19) implying resistant S.aureus traits were most common in hospital community. Apart from these, MTCC type strains did not exhibit any resistance towards the antibiotics tested. S8 exhibits intermediate resistance to erythromycin, oflaxin, streptomycin and tetracycline. Layer et al. (26) observed a similar findings working with S.aureus strains at German hospital and found that most of S.aureus isolates were resistant to penicillin, ciprofloxacin, clindamycin, erythromycin and gentamicin.
The present study emphasizes comparison of methods of identification of methicillin resistance S. aureus isolated from the burn wound site. Since, the time taken for the identification of antibiotic resistance strain and the related medication and mortality, demand a speedy and authenticated procedures. In the present study, microbial colonies isolated from burn wound site subjected to screening for MRSA and S. aureus using standard procedures and the MRSA strains were further authenticated by their phenotypical and genotypical methods followed by MALDI-TOF analysis. We found among 10 S. aureus isolates, seven isolates exhibited positive response to both the said methods and suggests both methods were suitable for identification of MRSA species. Though, analysis through MALDI-TOF was cost -intensive, however, the time taken for the analysis and the authentication further suggests, analysis through MALDI-TOF will certainly reduce the mortality in burn ward.
Acknowledgments
One of the authors Mr. E. Madhava Charyulu thanks Director, CLRI for providing the facilities to carry out the work.
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Primer
Primer sequences ( 5' - 3')
Amplification size ( bp)
